Viruses commandeer host membrane trafficking to promote viral replication. While this is a critical control point for virus infection, how viruses hijack host proteins and pathways to promote infection is incompletely understood. Viral-mediated alterations in membrane trafficking are best understood for building replication compartments and sites of assembly for RNA viruses. However, much less is known about how DNA viruses influence membrane remodeling in the host. Human cytomegalovirus (CMV) is a complex DNA virus and a member of the herpesvirus family that establishes a lifelong infection in the host. During infection, CMV induces alterations in membrane trafficking that results in increased biogenesis of multivesicular bodies (MVBs), organelles important for signaling and exocytosis or degradation of cargo. During infection, MVBs become loaded with virus particles and dense bodies (vesicles of viral tegument protein). The mechanisms by which CMV controls host membrane trafficking pathways, and particularly MVB biogenesis, are not understood. Therefore, defining the viral proteins and host targets important for the regulation of membrane trafficking is an important goal with important implications for host and virus biology. We have identified two CMV proteins, pUL135 and pUL136, which regulate MVB biogenesis. Recombinant viruses lacking UL135 or UL136 genes exhibit profound alterations in MVB biogenesis and the incorporation of viral cargo into MVB. Consistent with these phenotypes, we have identified host interacting proteins for both pUL135 and pUL136 proteins that are important for membrane trafficking and MVB biogenesis. The discovery of two viral proteins and their host interactions strongly position us to define the mechanisms by which CMV modulates membrane trafficking and MVB biogenesis. We hypothesize that UL135- and UL136-host interactions co-opt host membrane trafficking and MVB pathways to modulate virus replication. Three aims are proposed to address this hypothesis. We will define the association of UL135 and UL136 with subcellular membrane compartments and host proteins in Aim 1. In Aim 2, we will define the significance of host interactions to virus replication, membrane trafficking, and incorporation of cargo into MVBs to understand the mechanistic basis of CMV- mediated control of host trafficking. Finally, we will investigate the role of UL135 and UL136 and their host interacting partners in regulating MVB biogenesis and determine how this regulation impacts viral egress. Our studies will define the virus-host interactions and trafficking pathways targeted by CMV and their significance to infection. Furthermore, we anticipate that our findings will uncover common strategies used by many viruses to manipulate trafficking pathways.